This invention relates to a method for producing hydrophilic monomers, which are particularly useful for electrophoresis, and to electrophoresis compositions, more particularly, to an electrophoresis gel composition that is hydrolytically stable and has high resolution for biological macromolecule separations. The invention further relates to a polymer composition which effectively suppresses electroendoosmosis, is hydrolytically stable and has high resolution for use in capillary electrophoresis and microchannel-based separations of macromolecules. The invention also relates to the preparation of electrophoresis compositions, electrophoresis gels, and coating compositions. The invention further relates to the use of said compositions, gels and polymer media for high resolution electrophoretic separations of proteins, nucleic acids, and other biological macromolecules.
The publications and other materials used herein to illuminate the background of the invention and in particular, cases to provide additional details respecting the practice, are incorporated herein by reference.
Electrophoresis gels have been widely used for the separations of biological macromolecules such as proteins, nucleic acids, and the like. There are essentially two types of gels in use: agarose gels and polyacrylamide gels. Polyacrylamide gels, in general, have higher resolving power than agarose gels. Since gel casting is rather tedious and the quality of hand-cast gels is inconsistent, there is a need for precast, xe2x80x9cready to usexe2x80x9d gels. Generally, precast gels are manufactured and supplied in buffers of pH between 8 and 9. Under these conditions, precast agarose gels are stable, and have a shelf life of one year at 4xc2x0 C. However, precast polyacrylamide gels are unstable, and depending on use, have a shelf life of only three months at 4xc2x0 C. As precast polyacrylamide gels age in alkaline conditions (pH above 7), the electrophoretic mobility of biological macromolecules through these gels decreases and the separation resolution deteriorates. The short shelf life of precast polyacrylamide gels is primarily attributed to the hydrolytic degradation of acrylamide moieties in the gel, while the crosslinking units, usually N,Nxe2x80x2-methylene bisacrylamide, are relatively stable. Due to the short shelf life of precast polyacrylamide gels, it is difficult for a manufacturer to mass-produce and to store large quantities of gels, and it is inevitable that some customers have to throw away some unused but xe2x80x9cexpiredxe2x80x9d gels. Therefore, it is highly desirable to have a gel that has a similar resolution to polyacrylamide gel, but a longer shelf life. Since the manufacturing and application of precast polyacrylamide gels are well established, it is even more desirable to have a stable, high resolution gel system that can be manufactured and used in the same manner as polyacrylamide gels.
Recognizing the fact that the short shelf life of precast polyacrylamide gels is due to the hydrolytic degradation of acrylamide moieties in alkaline condition, Takeda et al. (U.S. Pat. No. 5,464,516), Engelhorn et al. (U.S. Pat. No. 5,578,180) and Bjellqvist et al. (WO 96/16724) developed neutral buffer systems to replace the conventionally used Tris-HCl buffer (pH=8.8) in sodium dodecyl sulfate (SDS) polyacrylamide gels to reportedly improve the shelf life of precast polyacrylamide gels. However, the gel running buffer has to be changed to be compatible with gel buffer, and the protein separation patterns that are obtained from these systems are different from traditional SDS polyacrylamide electrophoresis based on the Laemmli system (Laemmli, Nature 277:680-685 (1970)).
Several vinyl-based monomers were proposed to replace acrylamide in the standard polyacrylamide gel system in order to improve gel stability. Shorr and Jain (U.S. Pat.No. 5,055,517) disclosed the use of N-mono- or di-substituted acrylamide monomers, such as N,Nxe2x80x2-dimethylacrylamide (DMA), in electrophoresis gels. Although DMA is more stable than acrylamide, DMA is very hydrophobic and is useful in only a limited number of electrophoretic applications, such as for certain types of nucleic acid analyses.
Kozulic and Mosbach (U.S. Pat. No. 5,319,046) disclosed the use of N-acryloyl-tris-(hydroxymethyl)aminomethane (NAT), and Kozulic (U.S. Pat. No. 5,202,007) disclosed the use of sugar-based acrylamide derivatives in electrophoresis gels. Because of the presence of several hydroxyl groups in the monomers, these monomers are extremely hydrophilic. However, Chiari et al (Electrophoresis 15:177-186 (1994)) reported that NAT is less stable than acrylamide. On the basis of molecular modeling, Miertus et al (Electrophoresis 15:1104-1111 (1994)) concluded that, when there are two atoms between the amide linkage and the hydroxyl group (as is the case for NAT, sugar-based acrylamide derivatives, and N-(2-hydroxyethyl)acrylamide), the hydroxyl group facilitates the hydrolysis of amide linkages.
In a series of articles and a U.S. patent, Righetti et al. (U.S. Pat. No. 5,470,916; Electrophoresis 15:177-186 (1994); Electrophoresis 16:1815-1829 (1995)) disclosed the use of N-mono- and di-substituted hydroxyethoxyethyl-(meth)acrylamides and their analogs in electrophoresis gels. The formula of the monomers disclosed by Righetti et al. in these 
N-(Hydroxyethoxyethyl)acrylamide (HEEAA) was identified as the preferred monomer, because of its extreme hydrophilicity and resistance to alkaline hydrolysis.
However, Righetti et al. (WO 97/16462; Electrophoresis 17:723-731 (1996); Electrophoresis 17:732-737 (1996); Electrophoresis 17:738-743 (1996)) subsequently reported that the HEEAA monomer had a peculiar tendency to auto-polymerize during storage as a 50% aqueous solution at 4xc2x0 C., even in the presence of free radical inhibitor. In view of this auto-polymerization tendency of HEEAA, Righetti et al. disclosed in these references the use of N-mono- and di-substituted hydroxyalkyl-(meth)acrylamides as an alternative in electrophoresis gels. The formula of the monomers disclosed by Righetti et al. in these references is: 
N-(Hydroxypropyl)acrylamide (HPAA) was claimed by Righetti et al. to be extremely hydrophilic and resistant to alkaline hydrolysis. However, to applicants"" knowledge there have been no further reports on HPAA-based gels by Righetti""s group or other groups, and there have been no HPAA-based commercial products.
For capillary electrophoresis (CE) of biological molecules, linear non-crosslinked polymers are commonly used rather than crosslinked gels due to easy replacement of media between runs. Many water-soluble, non-ionic polymers were shown to have utility as sieving media for CE. These include polyacrylamide (J Chromatogr, 516:33-48 (1990)), substituted celluloses (U.S. Pat. No. 5,534,123), polyethylene oxide (J Chromatogr 781:315-25 (1997)), and polyacrylamide derivatives (U.S. Pat. Nos. 5,552,028 and 5,567,292) for capillary-based DNA sequencing. For CE of double-stranded DNA fragments, agarose (Electrophoresis 12:1059-1061 (1991)), polymers made from N-substituted acrylamide monomers (Electrophoresis 15:177-186 (1994); Electrophoresis 17:723-731 (1996); U.S. Pat. No. 5,470,916), polyvinyl alcohol, and polyvinylpyrrolidone (U.S. Pat. No. 5,089,111) are known. These suggest that any hydrophilic, non-charged polymer will have some utility in electrophoretic separation of biological molecules in capillaries (Electrophoresis 19:3114-3127 (1998)).
A problem in capillary electrophoresis (CE) is electroendoosmosis (EEO) which must be lo suppressed in order to obtain good resolution of analytes. Typically, the capillaries used in CE are fused silica glass. The silanol groups on the inner surface of capillaries will become negatively charged under the alkaline pH of separation buffers, and because these are fixed charges, when a high voltage is applied ( greater than 100 V/cm) the mobile solution in the capillary is pulled toward the cathode. This phenomenon, termed EEO, retards the migration of negatively charged analytes such as DNA and proteins, and obscures the ability of the system to accurately separate them by size. Therefore, it is necessary to neutralize or shield the charges on the capillary wall to suppress EEO. There are two methods to solve this problem: (1) to perform chemical modification of the capillary wall, and (2) to use polymers that adsorb to the capillary wall.
There are several known methods that apply chemical modification of the capillary glass surface to suppress EEO.
Hjertxc3xa9n (U.S. Pat. No. 4,680,201; J Chromatogr 347:191-198 (1998)) described a method in which he reacted the silanol groups on the capillary wall with a bifunctional silane reagent, such as methacryloylpropyl trimethoxysilane, to introduce vinyl groups on the capillary wall. A polyacrylamide layer is attached to it by polymerization of acrylamide to coat the wall. While this method is widely used, the coating degrades under the high temperature alkaline conditions used in electrophoresis due to hydrolysis of the Si-O-Si bonds. Also, the amide groups on polyacrylamide will undergo hydrolysis and result in charged moieties. Either of these will lead to a buildup of charge on the capillary wall, and will be observed as an increase in EEO and decrease in resolution.
Novotny et al. (U.S. Pat. Nos. 5,074,982, 5,143,753) described a partial solution to the problem by use of Grignard chemistry to introduce more stable anchoring sites for the vinyl groups on the wall by use of Si-C bonds, but this still did not address the instability of the amide groups on the polyacrylamide coating.
To overcome the hydrolysis of acrylamide, Righetti et al described the use of stable substituted acrylamide monomers as coating composition for the inner walls of capillaries in combination with the Grignard chemistry (U.S. Pat. No. 5,470,916 and WO97/16462). In this disclosure, Righetti reports that N-(2-hydroxyethyl)acrylamide (HEAA) is not useful due to instability (Electrophoresis 15:1104-1111 (1994)).
An alternative approach to the chemical treatment of capillaries is to use polymers which directly interact with or adsorb to the capillary wall, resulting in a transient xe2x80x9ccoatingxe2x80x9d or zone of high viscosity which suppresses EEO. This approach is less tedious and time-consuming compared to the preparation of chemically coated capillaries, and operationally less expensive because the capillaries do not need to be replaced as often as the chemically treated capillaries. The polymer solution could have a dual function, (1) as coating of capillary surfaces and (2) as media for effective molecular separation.
It was demonstrated by Gilges and coworkers that non-ionic polyvinylalcohol was useful for protein electrophoresis in bare fused silica capillaries (J High Resolution Chromatography 15:452-457 (1992)). Yeung and co-workers reported that even pre-washing a capillary with polyvinylpyrrolidone (PVP) gives enough adsorption of PVP to the capillary wall that it can be re-filled with another polymer solution useful for DNA sequencing (Anal Chem 70:4044-4053 (1998)). These two reports suggest that most hydrophilic non-ionic polymers can adsorb to bare silica surfaces and suppress EEO. In theory, such capillaries may be reused multiple times, provided that the coating can be washed away by refilling of polymer, or stripped off by acid treatment between uses, and thereby, fresh coating is regenerated for each use. In a practical sense, however, it is not easy to completely remove the coating between uses, and build up of hydrolytically unstable polymers on the capillary wall will become a problem as it will be the cause of increase in EEO.
Madabhushi et al (U.S. Pat. Nos. 5,552,028 and 5,567,292) reported that poly(N,N-dimethylacrylamide) is capable of suppressing EEO when injected into a bare fused-silica capillary, and that it can provide high performance in molecular separation for repeated use (U.S. Pat. Nos. 5,552,028 and 5,567,292). Poly(N,N-dimethylacrylamide) is now commercially available from PE Biosystems as the xe2x80x9cPOPxe2x80x9d matrix for use in DNA sequencing and fragment analyses in CE (Electrophoresis, 1998. 19, 224-230). The disadvantage of POP, however, is that it lacks resolution in the high molecular weight region and gives shorter read lengths in DNA sequencing compared to the classical linear polyacrylamide. Madabhushi et al also discloses N-substituted polyacrylamides, in which one possible substituent is hydroxyl-substituted C1 to C3 alkyl, as useful for this application, but are not specific as to which compounds are particularly useful. Furthermore, Madabhushi et al restricts the molecular weight of such polymers to be between 5,000 to 1,000,000 Daltons. Although Madabhushi et al relies heavily on the self-coating aspect of the polymer, they do not teach how to improve resolution.
Although N-(2-hydroxyethyl)acrylamide (HEAA) is an analog of the N-(hydroxyalkyl) acrylamides disclosed by Righetti (WO 97/16462), it has never been specifically reported as a monomer for electrophoresis gels. For example, Righetti specifically excludes HEAA in his patent applications and references. This is partially because HEAA was not commercially available, but more importantly, HEAA was believed to be unstable to hydrolysis, like N-acryloyl-tris-(hydroxymethyl)aminomethane (NAT) (Electrophoresis 15:1104-1111 (1994)).
Several preparation methods for HEAA have been reported in the literature. Saito et al (Macromolecules 29:313-319 (1996)) described a two-phase method for the preparation of HEAA. The organic phase contains acryloyl chloride and ethyl acetate solvent, and the aqueous phase contains sodium hydroxide and ethanolamine. The product is recovered from the organic phase, and further purified by silica gel chromatography. There are two inherent disadvantages with this method, however. First, HEAA is readily soluble in water, and ethyl acetate extraction is not efficient. Second, it is impractical to produce large quantities of HEAA by silica gel chromatography.
Chen (ACS Symposium Series 322:283-290 (1986)) disclosed a one-phase method in which acryloyl chloride was reacted with two equivalents of ethanolamine in acetonitrile. Although high-yield HEAA can be obtained in acetonitrile solution, no purification method was provided, other than removing acetonitrile by distillation. Removal of acetonitrile in this manner results in some polymerization of the HEAA monomer during purification.
Righetti et al (WO 97/16462; Electrophoresis 17:723-731 (1996)) disclosed another one-phase method for the preparation of N-(hydroxyalkyl)acrylamides. They reported that ethanol is the best solvent for this reaction. Since ethanol is reactive towards acryloyl chloride, the reaction has to be conducted between xe2x88x9230xc2x0 C. and xe2x88x9270xc2x0 C. Silica gel was also used for further purification.
Murashige and Fujimoto (JP 61-068454 and JP 61-000053) disclosed a method in which N-(hydroxyethyl)acrylamide was prepared by treating ethanolamine with C1-22 alkyl acrylate or acrylic acid. The monomer was directly converted to its polymer, and no monomer purification method was disclosed.
Jones (U.S. Pat. No. 2,593,888) describes the preparation of hydroxyalkyl(meth)acrylamide monomers in a reported high state of purity, as well as the polymerization of these monomers to produce water-soluble polymers. One monomer, HEAA, is prepared by reacting an excess of ethanolamine with acryloyl chloride in acetonitrile at a reduced temperature, followed by filtration and solvent removal.
Thus, there is a need to develop additional hydrophilic monomers for preparing electrophoresis compositions, and particularly electrophoresis gels having the combined properties of hydrolytic stability and high resolution. This need in the art is satisfied by the present invention, as described in further detail below.
There is also a need to develop additional linear polymers, particularly for capillary electrophoresis and microchannel-based separations, having the combined properties of suppressing electroendoosmosis, hydrolytic stability and high resolution. This need in the art is satisfied by the present invention, as described in further detail below.
There further is a need to develop a method for producing high purity N-(hydroxy-ethyl)acrylamide (BEAA) and similar hydrophilic monomers simply and on a large scale. This need is satisfied by the present invention, as described in further detail below.
It is an object of this invention to provide a method for the preparation of high-purity hydrophilic acrylamide or methacrylamide derivatives containing hydroxy groups simply and on a large scale.
It is also an object of this invention to provide compositions useful for electrophoresis, including pre-cast gels and monomer compositions for coating capillaries or for preparing electrophoresis gels or polymers, for the separation of biological macromolecules, such as proteins, nucleic acids and the like.
It is a further object of this invention to provide a gel composition for electrophoretic separations, which has combined high resolution and hydrolytic stability.
It is also an object of this invention to provide a linear polymer for capillary electrophoresis that effectively suppresses electroendoosmotic flow and at the same time gives high performance in electrophoretic separation of analytes in capillaries.
As used herein, the terms (meth)acryloyl chloride, (meth)acrylic acid and (meth)acrylamide are intended to refer to acryloyl or methacryloyl chloride, acrylic or methacrylic acid and acrylamide or methacrylamide, respectively.
According to one aspect of the present invention, hydrophilic monomers are produced by reacting (meth)acryloyl chloride with an aminoalcohol in a polar solvent, which favors amidation in the presence of a base. The reaction mixture is subjected to deionization, solvent removal and optionally, filtration. In one embodiment of the invention, water is added to the reaction mixture, the resulting aqueous solution is deionized, and the solvent is removed from the deionized aqueous solution to produce an aqueous solution of the hydrophilic acrylamide or methacrylamide derivatives containing hydroxy groups free of solvent. In a second embodiment of the invention, water is added to the reaction mixture, the solvent is removed from the resulting aqueous solution, and the resulting aqueous solution free of solvent is deionized to produce an aqueous solution of the hydrophilic acrylamide or methacrylamide derivatives containing hydroxy groups. In a third embodiment, the reaction mixture is first filtered to remove the salt byproduct prior to the addition of water for deionization or for the solvent removal step.
According to a second aspect of the invention, compositions useful for electrophoretic applications are provided. In one embodiment, these compositions may be used for coating capillary tubes used for capillary electrophoresis or to prepare a linear polymer as the sieving medium for capillary electrophoresis. In this embodiment, the composition comprises an aqueous solution of either the hydrophilic monomer (for coating) or the linear polymer (for use as a sieving medium). The linear polymer composition for use in capillary electrophoresis and microchannel-based separations of macromolecules effectively suppresses electroendoosmosis, is hydrolytically stable and has high resolution. In a second embodiment, the composition may also be used to prepare formulated solutions, which can be used to prepare precast gels or to prepare gels prior to use. Such gels are useful for DNA sequencing or other macromolecule separations. In this embodiment, the composition comprises a hydrophilic N-(hydroxyalkyl) (meth)acrylamide or N,N-di(hydroxyalkyl)(meth)acrylamide monomer, an optional comonomer, a bifunctional crosslinker, such as N,Nxe2x80x2-methylene bisacrylamide (BIS), a buffer and an optional denaturant. An initiator is added to effect the formation of the gel.
According to a third aspect of the invention, a stable, high-resolution electrophoresis gel is prepared by the free radical copolymerization of a hydrophilic monomer as described herein, preferably N-(2-hydroxyethyl)acrylamide (HEAA), an optional comonomer, and a bifunctional crosslinker, such as N,Nxe2x80x2-methylene bisacrylamide (BIS), in a buffer solution in a plastic or glass gel mold.